Genetic Variants in Vitamin-D Metabolism Genes (rs1155563, rs12785878 and rs10500804) among Females with Type-2 Diabetes Mellitus in Saudi Arabia

Background & Objective: Hypovitaminosis D has shown to be linked with T2DM development and control in numerous studies. The association of SNPs in genes related to VitD metabolism with T2DM has not been sufficiently studied. Consequently, our aim in the present study was to explore the association between genetic variants in genes connected with VitD, mainly a SNP in GC (rs1155563), a SNP in DHCR7 (rs12785878) and a SNP in CYP2R1 (rs10500804) with glycaemic parameters in females with T2DM in Saudi Arabia. Methods: The cross-sectional study included 149 females (age 38-52 years) with T2DM from Jeddah, Saudi Arabia (September 2022-March 2023). Blood was extracted from the participants for biochemical tests including measuring VitD [25(OH)D] concentration, parameters of glycaemia (HbA1c, insulin, fasting glucose and insulin sensitivity indices including HOMA2-IR and HOMA2-%β), and for genomic DNA isolation. Sanger DNA sequencing was used to screen for VitD genetic polymorphisms (rs1155563, rs12785878 and rs10500804). Results: Minor allele frequency for rs1155563C, rs12785878T and rs10500804G was 0.21, 0.23 and 0.37, respectively. Levels of 25(OH)D and glycaemic parameters as well did not show any significant difference between the genotypes of each SNP. Conclusion: This study showed lack of association of rs1155563 in GC, rs12785878 in DHCR7 and rs10500804 in CYP2R1 with VitD level primarily and with glycaemic parameters secondarily. Additional research is required to explore further other VitD genetic polymorphisms influencing T2DM which might lead consequently to genetically-based personalized management for T2DM.


INTRODUCTION
Several factors including environmental, nutritional, genetic facors can influence the development of Type-2 Diabetes Mellitus (T2DM) which is extremely prevalent in Saudi Arabia. 1 Among the important nutritional factors that appeared eminently in the research field is Vitamin-D (VitD).VitD deficiency has been found to be linked with T2DM in several studies. 2,3In addition, a number of studies have also shown that insufficient VitD level can rise the susceptibility of development of T2DM and VitD supplemental treatment can decrease the risk of development of T2DM or improve the glycemic control in T2DM patients. 4enetic factors can influence VitD level [which is mainly represented by level of serum 25-hydroxyvitamin-D (25(OH)D)] due to its high heritability (53%). 5It has been demonstrated in genome-wide association studies and candidate gene studies that single nucleotide polymorphisms (SNP) in VitD related genes have a substantial effect on 25(OH) D level. 6These genetic polymorphisms were evident in enzymes and proteins involved in VitD metabolic pathway including enzymes responsible for 25(OH)D activation [25-hydroxylase (CYP2R1) and 1-hydroxylase (CYP27B1)] and elimination [24-hydroxylase (CYP24A1)] and proteins for 25(OH) D transfer [VitD binding protein (GC)] and binding [vitamin-D receptor (VDR)].In addition to 7-dehydrocholesterol reductase (DHCR7) which facilitates the transformation of 25(OH)D to cholesterol. 7 large number of studies have shown significant link of certain SNPs in VDR and impaired insulin secretion and sensitivity as well as increased risk of T2DM. 7,8Only a few studies investigated SNPs in GC (the main carrier of VitD) in T2DM and found that these SNPs increase the risk of T2DM. 9 In regards with CYP2R1, only a single recent study conducted in Chinese population by Wang et al. 10 Has found an association between two SNPs in CYP2R1 and T2DM risk.The relationship between SNPs in Vitamin-D genes and T2DM has not been sufficiently addressed.Most of the studies focused mainly on the association between SNPs in VDR and T2DM. 11To our knowledge, little is known about genetic polymorphisms in VDBP (rs1155563), DHCR7 (rs12785878) and CYP2R1 (rs10500804) as new genetic markers for T2DM.A potentially vital question is whether genetic polymorphisms in genes linked to VitD metabolic process will associate with T2DM and impact its control accordingly.We aimed in the current research to study the frequency of SNPs in VitD interrelated genes, mainly rs1155563 in GC, rs12785878 in DHCR7 and rs10500804 in CYP2R1 and their link with measures of glycaemia (insulin sensitivity indices, fasting insulin, blood sugar and c-peptide) in females diagnosed with T2DM in Kingdom of Saudi Arabia.

METHODS
This study is a cross-sectional study that involved 149 females diagnosed with T2DM (age between 38 and 52 years) from Jeddah (the western area of Saudi Arabia), (September 2022-March 2023).SNPs in genes related to VitD metabolic pathway (specifically a SNP in GC (rs1155563), a SNP in DHCR7 (rs12785878) and a SNP in CYP2R1 (rs10500804) were assessed among the women in the study.The association of the genotypes of these SNPs with VitD [25(OH)D level] and parameters of glycaemia (HbA1c, insulin, fasting glucose, c-peptide and insulin sensitivity indices including HOMA2-IR and HOMA2-%β) was also investigated.Females who joined in the present study were referred to King Fahad Medical Research Centre (KFMRC), King Abdulaziz University (KAU), Jeddah, Saudi Arabia.All participants signed a printed detailed consent for partaking in this study.Ethical Approval: The study followed ethical principles of Declaration of Helsinki.Ethical approval for the study was taken from the Research Ethics Committee in Unit of Biomedical Ethics, Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University (KAU) (Ref No. 013-CEGMR-02-ETH; dated July 30, 2018).

Inclusion & Exclusion Criteria:
Participants in this study were selected according to particular inclusion and exclusion criteria.Women included in this study were formerly diagnosed with T2DM based on the recommendations of the American Diabetes Association 12 which diagnose patients with T2DM if fasting plasma glucose ≥7 mmol/L or HbA1c ≥48 mmol/mol.12Any woman having history of kidney or liver disease, rheumatoid arthritis, malabsorption syndrome, cancer, endocrinal disorder such as hyperthyroidism, hyperparathyroidism was excluded.In addition, any woman who reported any intake of medicines that have potential influence on VitD level (such as VitD, glucocorticoids and anticonvulsants) was also excluded Participants showing serum concentrations of creatinine and liver enzymes higher than the standard medical range were ruled out from the study (serum creatinine above 105µmol/L; Aminotransferase (AST) above 45 U/L; Alanine Aminotransferase (ALT) above 50 U/L and Alkaline Phosphatase (ALP) above 280 U/L) as well as participants with low thyroid stimulating hormone (TSH) levels (below 0.465 mIU/L).

Study process and blood biochemical tests:
Anthropometric measures were taken from all participants as well as blood samples that were taken and stored in free of additives (clot activators, anticoagulants, preservatives or separator material) tubes and tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA).Quantification of 25(OH)D, intact PTH, insulin and c-peptide levels in serum was performed through chemiluminescence immunoassay (CLIA), with a LIAISON auto-analyzer (DiaSorin Inc., Stillwater, MN, USA).The intraassay and inter-assay coefficient of variation (CV) for the analyzed samples were < 5%.Liver enzymes, creatinine, blood glucose, magnesium (Mg), calcium (Ca), phosphate (PO 4) , albumin and lipid profile were analyzed in serum through the colorimetric method utilizing a VITROS 250 Clinical Chemistry autoanalyzer (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA).The samples showed an intra-assay CV of 3.5% and an inter-assay CV of 3.9%.VitD deficiency was determined according to Institute of Medicine (IOM) recommendations. 13These guidelines define 25(OH)D concentration lower than 12 ng/ml as VitD deficiency, 25(OH)D level between 12 and 19 ng/ml as VitD insufficiency, and level from 20 to 50 ng/ml as VitD sufficiency.
Homeostasis Model Assessment 2 (HOMA2) was calculated in this study to evaluate insulin resistance (HOMA2-IR) and β-cell function (HOMA2-%β).HOMA2-IR and HOMA2-%β were estimated in a constant status from fasting glucose (3-25 mmol/L), fasting insulin (2.88-43.16mIU/L) and fasting c-peptide (0.6-10.5 µU/ml) with the aid of a PC HOMA calculator program (version 2.2.3) supplied by University of Oxford Diabetes Trials Unit, accessible at https://www.dtu.ox.ac.uk/homacalculator/.Fasting insulin and HOMA2 were not measured in women who were taking insulin as intake of exogenous insulin can affect these parameters.Genetic polymorphisms screening: Genomic DNA was isolated initially using DNA extraction kit (53104, Qiagen, Hilden, Germany).Assessment of the concentration and purity the DNA filtrate was done using NanoDrop spectrophotometer (ND-1000 UV-VIS).Screening for specific SNPs in genes related to VitD metabolism (rs1155563 in GC, rs12785878 in DHCR7 and rs10500804 in CYP2R1) was conducted.First, primers' designs for these SNPs were prepared with web-based Primer3 (v.0.4.

1) program (Table-I).
Relationship of Genetic Variants in Vitamin-D Metabolism Genes with T2DM

General and biochemical results of the included females are shown in Table-II. Median of VitD [25(OH)
D] level in the participating females with T2DM was 12 ng/ml.Based on IOM 21 VitD status guidelines, 50% had VitD deficiency, 32% had VitD insufficiency, and 18% had optimal VitD levels.

Shatha Matoug Alharazy
reference genotype TT and GG for rs1155563 and rs12785878, respectively.For rs10500804, 43% of the women had heterozygous TG and 41% had the reference genotype TT.It was also observed that minor allele frequency (MAF) for rs1155563C was 0.21.In addition, MAF for rs12785878T and rs10500804G was 0.23 and 0.37, respectively.VitD level and levels of glyaemic parameters among the subcategorized genotypes of rs1155563, rs12785878 and rs10500804 are shown in Table-III.The results showed no significant difference in levels of 25(OH)D, HbA1c, fasting glucose, fasting insulin, fasting C-peptide and insulin sensitivity indices including (HOMA2-IR and HOMA2-%β) between the genotypes of each SNP.In addition, there was no significant difference in bone parameters, lipid profile, LFT, creatinine and TFT between the three genotypes of the studies SNPs.

DISCUSSION
The current research described the allele frequency of three SNPs in genes engaged in VitD metabolic pathway in T2DM female population in Saudi Arabia.The MAF of the SNP rs1155563 in GC (the gene encoding VitD carrier protein), the SNP rs12785878 in DHCR7 (the gene encoding the enzyme needed for skin ViD synthesis) and the rs10500804 in CYP2R1 (the gene encoding the enzyme required for VitD activation in the liver) were all described in this study.The MAF of rs1155563 T/C was 0.21 which was identical to MAF seen in another study in Arabs, while MAF of

Table - I
: The design of PCR primers used for the selected SNPs in GC, DHR7 and CYP2R1 genes.
*Annealing PCR temperature.F is forward and R is reverse. ).

Table -
II: General and biochemical characteristics of the participating women.Table-III: Vitamin-D and glycaemic parameters levels in the different genotypes of the studied SNPs.